Methods and materials for treating inflammatory diseases

ABSTRACT

The invention provides methods and materials related to the treatment of inflammatory diseases such as rheumatoid arthritis. Specifically, the invention provides methods and materials for treating inflammation by reducing production of an inflammatory cytokine such as IFN-γ, IL-1β, and TNF-α. The invention also provides methods and materials for identifying reagents that can be used to treat inflammatory diseases. Specifically, the invention provides non-human animals containing human synovial tissue as well as methods for using such non-human animals to determine the influence of various test reagents on the inflamed state of human synovial tissue.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0001] Funding for the work described herein was provided by the federalgovernment, which may have certain rights in the invention.

BACKGROUND

[0002] 1. Technical Field

[0003] The invention relates to methods and materials involved in thetreatment of inflammatory disease such as rheumatoid arthritis. Inaddition, the invention relates to reagents that reduce inflammation aswell as animal models for identifying such reagents.

[0004] 2. Background Information

[0005] Rheumatoid arthritis (RA) is a systemic inflammatory disease thatpreferentially affects the synovial membrane and leads to irreversibledamage of cartilage and bone. CD4⁺ T cells are the dominant cell type inthe inflammatory infiltrates that together with the genetic associationto MHC class II molecules has been taken as evidence for a central roleof T cells in the pathogenesis of the disease (Harris E D Jr, W. B.Saunders (1997)). Tissue infiltrating CD4⁺ T cells display multipleproperties suggestive for local antigen recognition, such as expressionof IL-2 receptors and clonal proliferation in the joint (Goronzy J J andWeyand C M, Rheum. Dis. Clin. North Am. 21:655-675 (1995)). Furthersupport for in situ T cell activation has come from the structuralorganization of T and B cells in the inflamed synovia. In fact, T and Bcells have a tendency to form aggregates that morphologically andfunctionally resemble germinal centers (Kurosaka M and Ziff M, J. Exp.Med 158:1191-1210 (1983) and Schroder A E et al., Pro. Natl. Acad. Sci.U.S.A. 93:221-225 (1996)). Studies on T cell derived cytokines, however,have uniformly demonstrated that T cell products are particularly scarcein rheumatoid synovitis (Firestein G S and Zvaifler N J, ArthritisRheum. 33:768-773 (1990) and Feldman M et al., Cell 85:307-310 (1996)).The paucity of IL-2- and interferon-γ (IFN-γ)-producing T cells in thesynovial lesions has remained an enigma.

[0006] Interleukin-16 (IL-16₎, originally described as a lymphocytechemoattractant factor, is a natural ligand of the CD4 molecule (CenterD M and W Cruikshank, J. Immunol. 128:-2563-2568 (1982); Cruikshank W Wet al., J. Immunol. 146:2929-2934 (1991); Cruikshank W W et al., Proc.Natl. Acad. Sci. U.S.A. 91:5109-5113 (1994); and Ryan T C et al., J.Biol. Chem. 270:17081-17086 (1995)). In fact, IL-16 is apro-inflammatory cytokine that interacts with CD4 molecules and induceschemotaxis as well as IL-2 receptor and HLA-DR expression (Center D M etal., Int. J. Biochem. Cell Biol. 29:1231-1234 (1997)). Further, IL-16 issynthesized as a precursor protein, pro-IL-16, with CD8⁺, but not CD4⁺,T cells having the ability to secrete the bioactive form.

SUMMARY

[0007] The invention provides methods and materials for treatinginflammatory diseases. Specifically, the invention provides methods andmaterials that reduce expression of an inflammatory cytokine such asIFN-γ, IL-1β, and TNF-α within inflamed tissue. For example, IL-16polypeptide, IL-16-encoding nucleic acid, and/or an IL-16-mimickingmolecule can be administered to a host such that production of aninflammatory cytokine is reduced. Alternatively, cells such as synovialtissue-derived CD8⁺T cells can be administered to a host containinginflamed tissue such that production of an inflammatory cytokine isreduced. It is noted that the IL-16 polypeptide, IL-16-encoding nucleicacid, IL-16-mimicking molecule, and/or cells can be administered with animmunosuppressive cytokine polypeptide, immunosuppressivecytokine-encoding nucleic acid, immunosuppressive cytokine-mimickingmolecule, and/or cells expressing an immunosuppressive cytokine.Examples of immunosuppressive cytokines include, without limitation,TGF-β1, IL-4, and IL-10. The invention also provides methods andmaterials for identifying reagents that can be used to treatinflammatory diseases. Specifically, the invention provides non-humananimals containing human synovial tissue as well as methods for usingsuch non-human animals to determine the influence of various testreagents on the inflamed state of human synovial tissue.

[0008] One aspect of the invention features a method for treating aninflammatory disease (e.g., rheumatoid arthritis). The method includesidentifying a host having inflamed tissue, and administering apharmaceutically effective amount of an IL-16 polypeptide or anIL-16-mimicking molecule to the host under conditions such that theexpression of an inflammatory cytokine (e.g., IFN-γ, IL-1β, and TNF-α)in the region of the inflamed tissue is reduced. The host is in need ofan anti-inflammatory treatment and can be a mammal (e.g., a human). TheIL-16 polypeptide can be recombinant human IL-16, and theIL-16-mimicking molecule can be a recombinant HIV gp120 polypeptide. Themethod also can include administering, to the host, a polypeptide suchas TGF-β1, IL-4 and/or IL-10.

[0009] In another embodiment, the invention features a method fortreating an inflammatory disease (e.g., rheumatoid arthritis) in a host.The method includes providing nucleic acid to the host. The nucleic acidencodes an IL-16 polypeptide, and the host expresses the IL-16polypeptide from the nucleic acid such that the expression of aninflammatory cytokine (e.g., IFN-γ, IL-1β, and TNF-α) is reduced in thehost. The host can be a mammal (e.g., a human). The IL-16 polypeptidecan be human IL-16. The method also can include providing a secondnucleic acid to the host. The second nucleic acid encodes animmunosuppressive cytokine (e.g., TGF-β1, IL-4, or IL-10).

[0010] Another embodiment of the invention features a method fortreating an inflammatory disease (e.g., rheumatoid arthritis) in a host.The method includes administering, to the host, cells that reduce theexpression of an inflammatory cytokine (e.g., IFN-γ, IL-1β, and TNF-α).The host can be a mammal (e.g., a human). The cells can be, for example,synovial tissue-derived CD8⁺ T cells, cells that express IL-16 and/orTGF-α, and/or cells containing exogenous nucleic acid encoding an IL-16polypeptide. The IL-16 can be human IL-16. The cells can containexogenous nucleic acid that encodes a polypeptide such as TGF-β1, IL-4,and/or IL-10. In addition, the cells can have specificity for a synovialantigen, and can accumulate within synovial tissue.

[0011] In another aspect, the invention features a pharmaceuticalcomposition for treating an inflammatory disease (e.g., rheumatoidarthritis) in a host. The composition contains an IL-16 polypeptide(e.g., recombinant human IL-16) or an IL-16-mimicking molecule (e.g., arecombinant HIV gp120 polypeptide) and an immunosuppressive cytokine(e.g., TGF-β1, IL-4, and IL-10). The administration of the compositionto the host reduces the expression of an inflammatory cytokine (e.g.,IFN-γ, IL-1β, and TNF-α) in the host. The host can be a mammal (e.g., ahuman).

[0012] Another aspect of the invention features a non-human animalcontaining human synovial tissue. The non-human animal can be a murineanimal. In addition, the non-human animal can be immunocompromised. Forexample, the immunocompromised non-human animal can be a SCID mouse or arecombination-activating gene-deficient animal. At least a portion ofthe human synovial tissue can be located under the skin and/or in theback region of the non-human animal. The human synovial tissue can beinflamed human synovial tissue. The inflamed human synovial tissue canbe in a diffuse and/or follicular state of inflammation within theanimal. The human synovial tissue can be diffusely vascularized humansynovial tissue within the animal. The non-human animal can be aNOD/LtSz-Prkdc^(scid)/J mouse having human synovial tissue derived froma rheumatoid arthritis patient.

[0013] Another aspect of the invention features a method for identifyinga treatment reagent that reduces inflammation. The method includesadministering a test reagent to a non-human animal having human synovialtissue, at least a portion of the human synovial tissue being inflamed,and determining if the administration of the test reagent reduces theinflammation. A reduction in inflammation indicates that the testreagent is a treatment reagent. The reduction in inflammation can bedetermined by measuring IFN-γ production by the human synovial tissue.

[0014] Another aspect of the invention features an article ofmanufacture containing packaging material and an IL-16 polypeptide orIL-16-mimicking molecule. The packaging material contains a label orpackage insert indicating that the IL-16 polypeptide or IL-16-mimickingmolecule can be administered to a host for the purpose of treating aninflammatory disease.

[0015] In another embodiment, the invention features an article ofmanufacture containing packaging material and nucleic acid encoding anIL-16 polypeptide. The packaging material contains a label or packageinsert indicating that the nucleic acid can be administered to a hostfor the purpose of treating an inflammatory disease.

[0016] Another embodiment of the invention features an article ofmanufacture containing packaging material and cells that reduce theexpression of an inflammatory cytokine. The packaging material containsa label or package insert indicating that the cells can be administeredto a host for the purpose of treating an inflammatory disease.

[0017] Another aspect of the invention features the use of an IL-16polypeptide or an IL-16-mimicking molecule in the manufacture of amedicament for treating an inflammatory disease in a host in need of ananti-inflammatory treatment. The host has inflamed tissue, andadministering a pharmaceutically effective amount of the medicament tothe host reduces the expression of an inflammatory cytokine in theregion of the inflamed tissue.

[0018] In another embodiment, the invention features the use of anucleic acid in the manufacture of a medicament for treating aninflammatory disease in a host in need of an anti-inflammatorytreatment. The nucleic acid encodes an IL-16 polypeptide. The host hasinflamed tissue, and administering the medicament to the host reducesthe expression of an inflammatory cytokine in the region of the inflamedtissue.

[0019] Another embodiment of the invention features the use of cells inthe manufacture of a medicament for treating an inflammatory disease ina host in need of an anti-inflammatory treatment. The host has inflamedtissue, and administering the medicament to the host reduces theexpression of an inflammatory cytokine in the region of the inflamedtissue.

[0020] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

[0021] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

[0022]FIG. 1 contains three graphs depicting the level of cytokine mRNAproduction in human synovial tissue before and after implantation intosevere combined immune deficient (SCID) mice.

[0023]FIG. 2 contains three graphs depicting the level of cytokine mRNAproduction in human synovial tissue harvested from human synovium-SCIDmouse chimeras receiving either medium only (designated control) orautologous synovial tissue-derived T cells (designated adoptivetransfer).

[0024]FIG. 3 contains two graphs depicting the level of cytokine mRNAproduction in human synovial tissue harvested from human synovium-SCIDmouse chimeras receiving medium only, autologous CD3⁺/CD4⁺ T cells, orautologous CD3⁺/CD8⁺ T cells. Identical symbols represent data obtainedfrom tissue originating from the same patient.

[0025]FIG. 4 contains four bar graphs depicting the number of CD3⁺cells, CD68⁺ cells, IFN-γ-producing cells, and TNF-α-producing cells perhigh power field (hpf) in human synovial tissue harvested from humansynovium-SCID mouse chimeras receiving medium only or the designatedautologous synovial tissue-derived T cell population.

[0026]FIG. 5 is a bar graph depicting the level of IFN-γ mRNA productionin human synovial tissue harvested from human synovium-SCID mousechimeras receiving either medium only or autologous synovialtissue-derived T cells as well as either control antibody or anti-IL-16antibody.

[0027]FIG. 6 is a bar graph depicting the number of CD8⁺/IL-16⁺ andCD8⁻/IL-16⁺ cells per high power field in synovial tissue obtained fromsix RA patients.

[0028]FIG. 7 contains four bar graphs depicting the level of cytokineproduction in human synovial tissue harvested from human synovium-SCIDmouse chimeras receiving treatment with or without recombinant humanIL-16 (rhIL-16) for either ten or fourteen days.

[0029]FIG. 8 contains four bar graphs depicting the number of CD3⁺cells, CD68⁺ cells, IFN-γ-producing cells, and TNF-α-producing cells perhigh power field in human synovial tissue harvested from humansynovium-SCID mouse chimeras receiving treatment with or without rhIL-16for ten days.

DETAILED DESCRIPTION

[0030] The invention provides methods and materials related to thetreatment of inflammatory diseases. Specifically, the invention providesmethods and materials for treating inflammation by reducing productionof an inflammatory cytokine such as IFN-γ, IL-1β, and TNF-α. Clearly,reducing the level of an inflammatory cytokine within inflamed tissueprovides a useful means for treating inflammation. The invention alsoprovides methods and materials for identifying reagents that can be usedto treat inflammatory diseases. Specifically, the invention providesnon-human animals containing human synovial tissue as well as methodsfor using such non-human animals to determine the influence of varioustest reagents on the inflamed state of human synovial tissue. Non-humananimals containing human synovial tissue provide scientists with aninvaluable tool that allows for the identification of anti-inflammatoryreagents. For example, various test reagents can be administered to theliving non-human animals described herein, and the reagent's influenceon the state of inflammation within human tissue closely monitored.Thus, the non-human animals containing human synovial tissue describedherein provide scientists with an animal model that can lead to theidentification of countless different reagents that can be used to treatvarious forms of inflammation, including rheumatoid synovitis.

[0031] Methods and Materials for Treating Inflammation

[0032] Inflammatory diseases can be treated by administering apharmaceutically effective amount of an IL-16 polypeptide to a hostidentified as being in need of anti-inflammatory treatment such that theexpression of an inflammatory cytokine within inflamed tissue isreduced. The term “inflammatory disease” as used herein includes,without limitation, rheumatoid arthritis, inflammatory skin diseasessuch as psoriasis, inflammatory bowel diseases such as colitis, andinflammatory lung diseases such as asthma and bronchitis. Hosts in needof anti-inflammatory treatment can be identified using any medical ornon-medical technique that can identify the presence of inflamed tissue.Briefly, inflamed tissues are characterized by pain, swelling, redness,and heat. Such tissue can be detected by simple observation or by theuse of immuno-based diagnostic assays. For example, the presence andamount of molecules associated with inflammation such as inflammatorycytokines can be measured by enzyme-linked immunosorbant assay (ELISA)to determine if a particular tissue is inflamed. The term “inflammatorycytokine” as used herein includes, without limitation, a cytokine thatstimulates an inflammatory response. Examples of inflammatory cytokinesinclude, without limitation, IFN-γ, IL-1β, and TNF-α. The term “host” asused herein includes all animals capable of producing an inflammatoryresponse. Thus, the term “host” includes, without limitation, mammalssuch as mice, rats, rabbits, sheep, goats, cows, horses, monkeys, andhumans. It is noted that other molecules (e.g., anti-inflammatoryagents) and/or polypeptides (e.g., immunosuppressive cytokines) inaddition to IL-16 can be administered to a host such that production ofan inflammatory cytokine is reduced. The term “anti-inflammatory agent”as used herein refers to compounds that counteract or suppress theinflammatory process. Examples of anti-inflammatory agents include,without limitation, glucocorticoids, anti-rejection drugs such ascyclosporin, cytotoxic agents such as cyclophosphamide, anti-metabolitessuch as methotrexate and azathioprine, and TNF-α receptor and IL-1receptor antagonists. The term “immunosuppressive cytokine” as usedherein refers to cytokines that reduce a host's immune response.Examples of immunosuppressive cytokines include, without limitation,TGF-β1, IL-4, and IL-10.

[0033] The term “IL-16 polypeptide” as used herein refers to anypolypeptide that has an amino acid sequence similar to the amino acidsequence of recombinant human IL-16 (PeproTech, Rocky Hill, N.Y.)provided that that polypeptide can reduce production of an inflammatorycytokine within inflamed tissue. For example, such polypeptides can havethe amino acid sequence of human IL-16 (accession number 1945569), mouseIL-16 (accession numbers 2911795 and 3127043), or non-human primateIL-16 (accession numbers 3127033, 3127035, 3127037, 3127039, and3127041). It is important to note that the amino acid sequence of humanIL-16 can be altered provided the altered polypeptide retains theability to reduce production of an inflammatory cytokine. In otherwords, a wild-type human IL-16 polypeptide sequence can containmutations (e.g., deletions, insertions, and substitutions as well ascombinations thereof) provided the mutated form of IL-16 retains theability to reduce production of an inflammatory cytokine. Likewise, theamino acid sequence of other polypeptides such as TGF-β1, IL-4, andIL-10 can be used in a mutated form provided at least some function ofthe wild-type polypeptide is maintained. It will be understood thatIL-16 refers to the active form of the polypeptide, not the inactivepro-IL-16 form.

[0034] Nucleic acid that encodes a polypeptide having a wild-type oraltered amino acid sequence can be identified and obtained using anymethod. For example, nucleic acid encoding wild-type human IL-16 can bemutated using common molecular cloning techniques (e.g., site-directedmutageneses). Again, possible mutations include, without limitation,deletions, insertions, and substitutions, as well as combinations ofdeletions, insertions, and substitutions. In addition, nucleic acid andamino acid databases (e.g., GenBank®) can be used to identify wild-typeor altered amino acid sequences. Further, PCR and nucleic acidhybridization techniques can be used to identify nucleic acid encodingpolypeptides having wild-type or altered amino acid sequences. Briefly,any nucleic acid can be used as a probe to identify a similar nucleicacid by hybridization under conditions of low to high stringency. Suchsimilar nucleic acid then can be isolated, sequenced, and analyzed todetermine the degree of alteration from a wild-type sequence.

[0035] In general, high stringency conditions can be used to identifynucleic acid having a high degree of homology to a probe. Highstringency conditions can include the use of a denaturing agent such asformamide during hybridization, e.g., 50% formamide with 0.1% bovineserum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/S0 mM sodiumphosphate buffer at pH 6.5 with 750 mM NaCl, and 75 mM sodium citrate at42° C. Another example is the use of 50% formamide, 5× SSC (0.75 M NaCl,0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.5), 0.1% sodiumpyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50μg/mL), 0.1% sodium lauryl sulfate (SDS), and 10% dextran sulfate at 42°C., with washes at 42° C. in 0.2× SSC and 0.1% SDS. Alternatively, lowionic strength and high temperature can be used, for washing, forexample, 0.1×SSC (0.015 M NaCl/0.0015 M sodium citrate), 0.1% SDS at 65°C.

[0036] Moderate stringency conditions can be used to identify nucleicacid having a moderate degree of homology to a probe. Moderatestringency conditions can include the use of higher ionic strengthand/or lower temperatures for washing of the hybridization membrane,compared to the ionic strength and temperatures used for high stringencyhybridization. For example, a wash solution of 4×SSC (0.06 M NaCl/0.006M sodium citrate), 0.1% SDS can be used at 50° C., with a last wash in1×SSC at 65° C. Alternatively, a hybridization wash in 1×SSC at 37° C.can be used.

[0037] Low stringency conditions can be used to identify nucleic acidhaving a low degree of homology to a probe. Low stringency conditionscan include the use of higher ionic strength and/or lower temperaturesfor washing of the hybridization membrane, compared to the ionicstrength and temperatures used for moderate stringency hybridization.For example, a wash solution of 4×SSC (0.06 M NaCl/0.006 M sodiumcitrate), 0.1% SDS can be used at 37° C., with a last wash in 1×SSC at45° C. Alternatively, a hybridization wash in 2×SSC at 37° C. can beused.

[0038] Hybridization can be done by Southern or Northern analysis toidentify a DNA or RNA sequence, respectively, that hybridizes to aprobe. The probe can be labeled with a radioisotope such as ³²P, anenzyme, digoxygenin, or by biotinylation. The DNA or RNA to be analyzedcan be electrophoretically separated on an agarose or polyacrylamidegel, transferred to nitrocellulose, nylon, or other suitable membrane,and hybridized with the probe using standard techniques well known inthe art such as those described in sections 7.39-7.52 of Sambrook etal., (1989) Molecular Cloning, second edition, Cold Spring harborLaboratory, Plainview, N.Y. Typically, a probe is at least about 20nucleotides in length. In addition, probes longer or shorter than 20nucleotides can be used.

[0039] Inflammatory diseases also can be treated by administering apharmaceutically effective amount of an IL-16-mimicking molecule to ahost identified as being in need of anti-inflammatory treatment suchthat the expression of an inflammatory cytokine within inflamed tissueis reduced. The term “IL-16-mimicking molecule” as used herein includesmolecules that interact with CD4 and reduce production of aninflammatory cytokine. IL-16-mimicking molecules do not include IL-16polypeptides themselves, MHC molecules, anti-CD4 antibodies, ornaturally-occurring virus particles. Thus, recombinant polypeptidesseparated from virus particles can be IL-16-mimicking molecules. Forexample, recombinant HIV gp120 polypeptides can be IL-16-mimickingmolecules. Again, other molecules (e.g., anti-inflammatory drugs) andpolypeptides (e.g., immunosuppressive cytokines) in addition to anIL-16-mimicking molecule can be administered to a host such thatproduction of an inflammatory cytokine is reduced.

[0040] Potential IL-16-mimicking molecules such as recombinant HIV gp120polypeptides can be tested for the ability to specifically interact withCD4 polypeptide using methods well known in the art including, withoutlimitation, binding assays. In addition, potential IL-16-mimickingmolecules can be tested for the ability to reduce production of aninflammatory cytokine using various common methods such as RT-PCR andELISA. When assessing the ability of an IL-16-mimicking molecule toreduce production of an inflammatory cytokine, an IL-16 polypeptide canbe used as a positive control. For example, an IL-16 polypeptide and arecombinant HIV gp120 polypeptide can be used in parallel experiments toassess cytokine production within inflamed tissue.

[0041] Inflammatory diseases can be treated by providing nucleic acidthat encodes an IL-16 polypeptide to a host such that IL-16 polypeptideis expressed and the production of an inflammatory cytokine withininflamed tissue is reduced. Examples of nucleic acid that encodes IL-16polypeptides include, without limitation, human IL-16 (accession numberM90391), mouse IL-16 (accession numbers AF006001 and AF017111), andnon-human primate IL-16 (accession numbers AF017106, AF017107, AF017108,AF017109, and AF017110). Again, the amino acid sequence of an IL-16polypeptide can be mutated with respect to a wild-type IL-16polypeptide. Thus, the nucleic acid encoding an IL-16 polypeptide alsocan contain mutations as described herein. In addition, other molecules(e.g., anti-inflammatory drugs) and polypeptides (e.g., IL-16 andimmunosuppressive cytokines) in addition to an IL-16-encoding nucleicacid can be administered to a host such that production of aninflammatory cytokine is reduced. It is noted that the provided nucleicacid would be considered exogenous to the cells within the hostreceiving the nucleic acid.

[0042] The term “nucleic acid” as used herein encompasses both RNA andDNA, including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid can be thesense strand or the antisense strand. In addition, nucleic acid can becircular or linear.

[0043] The term “exogenous” as used herein with reference to nucleicacid and a particular cell refers to any nucleic acid that does notoriginate from that particular cell as found in nature. Thus, allnon-naturally-occurring nucleic acids are considered to be exogenous toa cell once introduced into the cell. It is important to note thatnon-naturally-occurring nucleic acid can contain nucleic acid sequencesor fragments of nucleic acid sequences that are found in nature providedthe nucleic acid as a whole does not exist in nature. For example, anucleic acid containing a genomic DNA sequence within an expressionvector is considered to be a non-naturally-occurring nucleic acid, andthus is considered to be exogenous to a cell once introduced into thecell, since that nucleic acid as a whole (genomic DNA plus vector DNA)does not exist in nature. Thus, any vector, autonomously replicatingplasmid, or virus (e.g., retrovirus, adenovirus, or herpes virus) that,as a whole, does not exist in nature is considered to be anon-naturally-occurring nucleic acid. It follows that genomic DNAfragments produced by PCR or restriction endonuclease treatment as wellas cDNA's are considered to be non-naturally-occurring nucleic acidsince they exist as separate molecules not found in nature. It alsofollows that any nucleic acid containing a promoter sequence andpolypeptide-encoding sequence (e.g., cDNA or genomic DNA) in anarrangement not found in nature is considered to be anon-naturally-occurring nucleic acid.

[0044] It is also important to note that a nucleic acid that isnaturally-occurring can be exogenous to a particular cell. For example,an entire chromosome isolated from a cell of person X would beconsidered an exogenous nucleic acid with respect to a cell of person Yonce that chromosome is introduced into Y's cell.

[0045] Nucleic acid encoding IL-16 or any other polypeptide can beobtained using common molecular cloning or chemical nucleic acidsynthesis procedures and techniques, including PCR. PCR refers to aprocedure or technique in which target nucleic acid is amplified in amanner similar to that described in U.S. Pat. No. 4,683,195, andsubsequent modifications of the procedure described therein. Generally,sequence information from the ends of the region of interest or beyondare used to design oligonucleotide primers that are identical or similarin sequence to opposite strands of a potential template to be amplified.Using PCR, a nucleic acid sequence can be amplified from RNA or DNA. Forexample, a nucleic acid sequence can be isolated by PCR amplificationfrom total cellular RNA, total genomic DNA, and cDNA as well as frombacteriophage sequences, plasmid sequences, viral sequences, and thelike. When using RNA as a source of template, reverse transcriptase canbe used to synthesize complimentary DNA strands. In addition, standardnucleic acid sequencing techniques and software programs that translatenucleic acid sequences into amino acid sequences based on the geneticcode can be used to determine whether or not a particular nucleic acidhas a nucleic acid sequence that encodes a particular amino acidsequence such as the amino acid sequence of IL-16.

[0046] Any method can be used to provide a host with nucleic acid. Infact, many methods for introducing nucleic acid into cells in vivo arewell known to those skilled in the art. For example, lipofection andviral-mediated nucleic acid transfer are common methods for introducingnucleic acid into a host's cells. In addition, naked DNA can bedelivered directly to cells in vivo as describe elsewhere (U.S. Pat. No.5,580,859 and U.S. Pat. No. 5,589,466 including continuations thereof).

[0047] Inflammatory diseases can be treated by administering cells to ahost identified as being in need of anti-inflammatory treatment suchthat the expression of an inflammatory cytokine within inflamed tissueis reduced. Cells that can reduce production of an inflammatory cytokineinclude, without limitation, cells that produce an IL-16 polypeptide.Such cells include, but are not limited to, CD8⁺/IL-16⁺ T cells as wellas any cell containing an exogenous nucleic acid that encodes andexpresses an IL-16 polypeptide. For example, synovial tissue-derivedCD8⁺ T cells that produce an IL-16 polypeptide can be administered to ahost such that the level of an inflammatory cytokine produced withininflamed tissue is reduced. In addition, cells producing an IL-16polypeptide can have specificity for a synovial antigen, and canaccumulate within synovial tissue. Further, IL-16-producing cells canexpress other polypeptides in addition to IL-16. For example, IL-16⁺cells can express TGF-β1, IL-4, and/or IL-10. The expression of an IL-16polypeptide as well as the expression of any additional polypeptide canbe from either endogenous nucleic acid or exogenous nucleic acid thatwas introduced into the cell. Moreover, other molecules (e.g.,anti-inflammatory agents) and/or polypeptides (e.g., immunosuppressivecytokines) can be administered to a host in addition to IL-16-producingcells.

[0048] As described herein, cells that reduce production of aninflammatory cytokine within inflamed tissue can express an IL-16polypeptide along with other polypeptides (e.g., IL-4, IL-10, andTGF-β1). Any method can be used to identify such cells. For example,immunohistochemistry and biochemical techniques can be used to determineif a cell expresses an IL-16 polypeptide. In addition, any method can beuse to make a cell express a foreign polypeptide or a polypeptide thatis not normally expressed by that particular cell type. Such methodsinclude, without limitation, constructing a nucleic acid such that aregulatory element promotes expression of a nucleic acid sequence thatencodes a polypeptide. Typically, regulatory elements are DNA sequencesthat regulate expression of other DNA sequences at the level oftranscription. Thus, regulatory elements include, without limitation,promoters, enhancers, and the like. Methods of identifying cells thatexpress an amino acid sequence from an exogenous nucleic acid also arewell known to those skilled in the art. Such methods include, withoutlimitation, immunocytochemistry, Northern analysis, and RT-PCR.

[0049] A cell containing exogenous nucleic acid can maintain thatexogenous nucleic acid in any form within the cell. For example,exogenous nucleic acid can be integrated into the genome of a cell ormaintained in an episomal state. In other words, a cell used accordingto the invention can be a stable or transient transformant.

[0050] Any method can be used to introduce an exogenous nucleic acidinto a cell. In fact, many methods for introducing nucleic acid intocells, whether in vivo or in vitro, are well known to those skilled inthe art. For example, calcium phosphate precipitation, electroporation,heat shock, lipofection, microinjection, and viral-mediated nucleic acidtransfer are common methods for introducing nucleic acid into cells. Inaddition, naked DNA can be delivered directly to cells in vivo asdescribe elsewhere (U.S. Pat. No. 5,580,859 and U.S. Pat. No. 5,589,466including continuations thereof). Further, nucleic acid can beintroduced into cells by generating transgenic animals. Thus, atransgenic animal can be made to express human IL-16 so that cells fromthat transgenic animal can be isolated and administered to host. Theadministration of such cells can reduce production of an inflammatorycytokine within inflamed tissue of the host. It is noted that an IL-16polypeptide can be isolated from transgenic animals expressing an IL-16transgene and used as described herein. For example, a transgenic farmanimal can be made to secrete IL-16 polypeptide into its milk such thatlarge amounts can be isolated. See, e.g., Mammary Gland Transgenesis:Therapeutic Protein Production (Fidel O. Castro and Juhani Janne eds.,Springer-Verlag 1997).

[0051] Transgenic animals can be aquatic animals (such as fish, sharks,dolphin, and the like), farm animals (such as pigs, goats, sheep, cows,horses, rabbits, and the like), rodents (such as rats, guinea pigs,mice, and the like), non-human primates (such as baboon, monkeys,chimpanzees, and the like), and domestic animals (such as dogs, cats,and the like). Several techniques known in the art can be used tointroduce nucleic acid into animals to produce the founder lines oftransgenic animals. Such techniques include, but are not limited to,pronuclear microinjection (U.S. Pat. No. 4,873,191); retrovirus mediatedgene transfer into germ lines (Van der Putten et al., Proc. Nail. Acad.Sci., USA, 82:6148-6152 (1985)); gene transfection into embryonic stemcells (Gossler A et al., Proc Natl Acad Sci USA 83:9065-9069 (1986));gene targeting into embryonic stem cells (Thompson et al., Cell,56:313-321 (1989)); nuclear transfer of somatic nuclei (Schnieke A E etal., Science 278:2130-2133 (1997)); and electroporation of embryos.

[0052] For a review of techniques that can be used to generate andassess transgenic animals, skilled artisans can consult Gordon (Intl.Rev. Cytol., 115:171-229 (1989)), and may obtain additional guidancefrom, for example: Hogan et al, Manipulating the Mouse Embryo (ColdSpring Harbor Press, Cold Spring Harbor, N.Y. 1986); Krimpenfort et al.,Bio/Technology, 9:844-847 (1991); Palmiter et al., Cell, 41:343-345(1985); Kraemer et al., Genetic Manipulation of the Early MammalianEmbryo (Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1985); Hammeret al., Nature, 315:680-683 (1985); Purscel et al., Science,244:1281-1288 (1986); Wagner et al., U.S. Pat. No. 5,175,385; andKrimpenfort et al., U.S. Pat. No. 5,175,384.

[0053] A pharmaceutically effective amount of any of the materialsdescribed herein (e.g., an IL-16 polypeptide, IL-16-mimicking molecule,IL-16-encoding nucleic acid, and IL-16-producing cell) refers to anyamount that does not cause significant toxicity to the host and reducesproduction of an inflammatory cytokine within inflamed tissue. Such anamount can be determined by assessing the clinical symptoms associatedwith the inflamed tissue before and after administering a fixed amountof a particular material (e.g., an IL-16 polypeptide). In addition, thepharmaceutically effective amount of a particular material administeredto a host can be adjusted according to the host's response and desiredoutcomes. Significant toxicity can vary for each particular patient anddepends on multiple factors including, without limitation, the patient'sdegree of inflammation, age, and tolerance to pain.

[0054] In addition, any of the materials described herein (e.g., anIL-16 polypeptide, IL-16-mimicking molecule, IL-16-encoding nucleicacid, and IL-16-producing cell) can be administered to any part of thehost's body including, without limitation, the joints, blood stream,lungs, intestines, muscle tissues, skin, peritoneal cavity, and thelike. Thus, an IL-16 polypeptide can be administered by intravenous,intraperitoneal, intramuscular, subcutaneous, intrathecal, andintradermal injection, by oral administration, by inhalation, or bygradual perfusion over time. For example, an aerosol preparationcontaining an IL-16 polypeptide can be given to a patient by inhalationto treat, for example, lung inflammatory diseases such as asthma andbronchitis. It is noted that the duration of treatment with any of thematerials described herein can be any length of time from as short asone day to as long as a lifetime (e.g., many years). For example, anIL-16 polypeptide can be administered at some frequency over a period often years. It is also noted that the frequency of treatment can bevariable. For example, an IL-16 polypeptide can be administered once (ortwice, three times, etc.) daily, weekly, monthly, or yearly.

[0055] Preparations for administration can include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents include, without limitation, propylene glycol,polyethylene glycol, vegetable oils, and injectable organic esters.Aqueous carriers include, without limitation, water as well as alcohol,saline, and buffered solutions. Preservatives, flavorings, and otheradditives such as, for example, antimicrobials, anti-oxidants, chelatingagents, inert gases, and the like may also be present.

[0056] Any method can be used to determine if the expression of aninflammatory cytokine within inflamed tissue was reduced. For example,the level of expression of a particular cytokine can be determined bymeasuring mRNA or polypeptide levels. Thus, the amount of aninflammatory cytokine within a tissue sample can be measured usingRT-PCR, functional assays (e.g., cell proliferation assays), orimmunological assays (e.g., ELISA). Moreover, clinical methods that canassess the degree of inflammation can be used to determine whetherinflammation in general is reduced. It is important to note that for thepurpose of this invention any reduction in the degree of inflammationmeans that the expression of at least one inflammatory cytokine, whethercurrently identified or not, has been reduced.

[0057] Methods and Materials for Identifying Treatment Reagents

[0058] The invention provides non-human animals that contain humansynovial tissue. Such non-human animals can maintain human synovialtissue in an inflamed state. Specifically, the human synovial tissue canremain inflamed and become diffusely vascularized after transplantationinto a non-human animal such as an immunocompromised mouse (e.g., aNOD/LtSz-Prkdcscid/J mouse). It is important to note that theinflammation observed within human synovial tissue obtained from humanpatients with RA is normally follicular or diffuse in nature. Follicularinflammation is characterized by the presence of T and B cells as wellas dendritic cells. In addition, tissue that is inflamed in a follicularfashion has a characteristic structure that resembles the germinalcenters of lymph nodes. On the other hand, diffuse inflammation ischaracterized by a diffuse infiltrate of T cells and macrophages. Thediffusely inflamed tissue has no real structural characteristics at thecellular level. Although not limited to any particular mode of action,it is noted that the human synovial tissue remains inflamed severalmonths following transplantation into an immunocompromised mouse.Presumably, this means that the specific antigens and cells mediatinginflammation in rheumatoid arthritis remain present and functionalwithin the transferred synovial tissue.

[0059] The term “non-human animal” as used herein refers to all animalsbut humans. Thus, a non-human animal can be an aquatic animal (such as afish, shark, dolphin, and the like), farm animal (such as a pig, goat,sheep, cow, horse, rabbit, and the like), rodent (such as a rat, guineapig, mouse, and the like), non-human primate (such as a baboon, monkey,chimpanzee, and the like), or domestic animal (such as a dog, cat, andthe like). Human synovial tissue can be obtained from human RA patientsby synovial biopsy or joint surgery. See, e.g., Kalunian et al.Arthroscopy in “Arthritis and Allied Conditions” W. Koopman (Ed.)Williams and Wilkins 1997, p.103-114.

[0060] Once obtained, the human synovial tissue can be implanted into anon-human animal. Typically, immunocompromised mice are used as thenon-human animals. For example, human synovial tissue is typicallyimplanted into RAG (recombination-activating gene)-deficient or SCIDmice. Normally, the human synovial tissue is implanted under the skin inthe back region of the non-human animal, although any location ispossible. Briefly, human synovial tissue can be cut macroscopically intopieces about three to four mm in size (e.g., about 30-60 cubic mm). Thecut tissue pieces then can be implanted into immunocompromised mice suchas NOD/Lt Sz-Prkdc^(scid)/J mice. It is noted that immunocompromisedmice typically are housed in special facilities to protect them fromexcessive exposure to pathogens. To implant the human tissue, the micecan be anesthetized. Any method can be used to anesthetize mice, forexample, methoxyflurane inhalation can be used together with anintraperitoneal administration of pentobarbital (about 50 mg/kg). Formethoxyflurane inhalation, cotton lightly soaked with methoxyflurane canbe placed into a syringe (e.g., a 10 cc syringe) lacking a plunger. Themouse's head then can be placed into the open end of the syringe and itsbreathing monitored. Typically, the mice are ready to use within five toten minutes of constant application of methoxyflurane. Onceanesthetized, the surgical area (e.g., the back region) is shaved, and asmall incision is made in the skin. From this incision, a canal can beprepared under the skin by, for example, gently sliding scissors underthe skin. After placing the human synovial tissue into the preparedcanal, the incision is sutured and methoxyflurane inhalation removed.Typically, the mice recover within five to ten minutes of methoxyfluraneremoval. After implantation, the human synovial tissue can becomediffusely vascularized within about seven days, and remain inflamed formany months.

[0061] The non-human animals containing human synovial tissue describedherein can be used to identify treatment reagents that reduce theinflammation observed within the human synovial tissue. Specifically, atest reagent can be administered to a non-human animal having humansynovial tissue, and the human synovial tissue, at least a portion ofwhich is inflamed, can be analyzed or monitored to determine if the testreagent reduced inflammation. Test reagents can be any type of material.For example, test reagents can be, without limitation, cells,polypeptides, lipids, amino acids, nucleic acids, drugs, and chemicalcompounds. In addition, test reagents can be administered to any part ofthe non-human animal's body. For example, a test reagent can be givenorally, nasally, intravenously, intraperitoneally, intramuscularly,subcutaneously, intrathecally, or intradermally. In addition, a testreagent can be directly administered to the inflamed human synovialtissue within the non-human animal. It is noted that a test reagent canbe administered at any dose, for any duration, and at any frequency.Further, any method can be used to determine if a particular testreagent reduced inflammation. For example, the production of aninflammatory cytokine within the inflamed tissue can be measured todetermine whether the inflammatory state of the human synovial tissuehas been reduced.

[0062] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES Example 1 Generation of Non-Human Animal Containing HumanSynovium (Human Svnovium-SCID Mouse Chimeras)

[0063] 1. Study Population and Source of Human Synovial Tissue

[0064] Synovial tissue from synovial biopsy or joint surgery wasobtained from eleven patients with active RA. Each patient fulfilled theACR 1987 revised criteria for RA (Arnett F. C., et al., Arthritis Rheum.31:315-324 (1988)). The mean age was 60 years (range 41-75 years) andthe mean disease duration was 23 years (range 4-51 years). All patientshad active synovitis at the time of tissue collection, and histology ofthe synovial tissue revealed dense mononuclear cell infiltrates. Sevenpatients had rheumatoid factor. Four of the eleven patients had beentreated with only nonsteroidal anti-inflammatory drugs (NSAIDs) for atleast three months prior to surgery. The remaining seven patients weretreated with low-dose corticosteroids (n=4), hydroxychloroquine (n=2),and/or methotrexate (n=3). All patients were typed for their HLA-DRB1alleles by PCR and subsequent oligonucleotide hybridization (BiotestDiagnostics, Danville, N.J.). Eight patients contained thedisease-associated allele designated HLA-DRB1*04, while two patientscontained the HLA-DRB 1*01 allele.

[0065] 2. Generation of Human Synovium-SCID Mouse Chimeras

[0066] To generate an in vivo model for human rheumatoid synovitis,human synovial tissue was collected and implanted into immunocompromisedmice. In preliminary studies, the recipient mouse strain, the size ofthe graft, and the implantation site were found to be critical inproviding optimal conditions for the persistence of inflammatory lesionswithin the engrafted tissue. When synovial tissue from a patient withactive synovitis was engrafted subcutaneously in the lower back regionof NOD-SCID mice, synovitis persisted at least two months. Fullengraftment of the tissue was usually achieved five to seven daysfollowing transplantation, and was associated with the formation of ahighly vascularized granulation tissue around the graft.

[0067] Briefly, six to eight week old NOD/LtSz-Prkdc^(scid)/J mice(NOD-SCID; mouse weight about 20-30 g; Jackson Laboratory, Bar Harbor,Me.) were anesthetized with pentobarbital (about 50 mg/kg; AbbottLaboratories, North Chicago, Ill.) intraperitoneally (i.p.) andmethoxyflurane (Mallinckrodt Veterinary, Mudelein, Ill.) by inhalation.These mice were housed in a barrier facility to protect them fromexcessive pathogen exposure. Before injection, the pentobarbital wasdiluted 1:10 in sterile PBS with about 300 μL being administered permouse. Extra pentobarbital was prepared in case an additionaladministration was needed. For methoxyflurane inhalation, cotton lightlysoaked with methoxyflurane was placed into a 10 cc syringe lacking theplunger while operating under a hood to avoid methoxyflurane inhalation.The mouse's head then was placed into the open end of the syringe andits breathing monitored. Care was taken to ensure that the mice were notover sedated with methoxyflurane since it can be toxic. Typically, micewere ready to use within five to ten minutes of constant application ofmethoxyflurane. Once anesthetized, each mouse was placed on its side onsterile gauze or paper towels, and its back region shaved withoutcutting the skin using sharp scissors. Care was taken to prevent themice from breathing hair shavings. Once a mouse was shaved, curvedforceps were used to pick up the skin and a small incision was madealong the dorsal midline. Once cut, one end of the skin was lifted andscissors were slid under the skin to prepare a canal for the implantedtissue. The inflamed human synovial tissue obtained from RA patients wasmacroscopically cut into equal pieces of about three to four mm in size(about 30-60 cubic mm) and placed in the prepared canal using straightforceps. Curved forceps were used to hold the tissue in place from theoutside while the incision was sutured. After removal of themethoxyflurane inhalation, the mice were monitored to ensure recovery.Typically, the mice recovered within five to ten minutes followingmethoxyflurane removal.

[0068] Engraftnent of the implanted human synovial tissue generallyoccurred within seven days. Mice were sacrificed after three weeks, andthe synovial tissue was retrieved and embedded in OCT compound(Tissue-Tek, Sakura Finetek, Torrance, Calif.) or shock frozen in liquidnitrogen for future RNA isolation.

[0069] Synovial tissue from eleven RA patients was implanted intoNOD/LtSz-Prkdc^(scid)/J mice. Histomorphology of the human tissue uponexplantation revealed that the inflammatory lesions persisted at leastthree to four weeks. In addition, the number of tissue infiltrating Tcells declined initially before reaching a plateau. Before implantation,fresh surgical synovial tissue exhibited a low level of IFN-γtranscription (median copy numbers of 33 per 2×10⁶ β-actin sequences).In addition, the macrophage products IL-1β and TNF-α were detected withmedian copy numbers of 186 and 295, respectively, per 2×10⁶ β-actinsequences before implantation. After engraftment intoNOD/LtSz-Prkdc^(scid)/J mice, the human synovial tissue exhibited anelevated level of mRNA synthesis for all three cytokines (FIG. 1).Specifically, two to three weeks after implantation, in situtranscription of IFN-γ, IL-1β, and TNF-α reached median copy numbers of492, 2580, and 14228, respectively, per 2×10⁶ β-actin sequences. The tento fifty fold increase in the production of cytokine transcripts in thexenografts was statistically significant (IFN-γ, p=0.002; IL-1β,p=0.002; TNF-α, p=0.01).

[0070] The kinetics of enhanced cytokine mRNA synthesis inxenotransplanted rheumatoid synovium were examined by implanting four toeight different mice with fragments of synovial tissue from the samedonor. Grafts were successively harvested between one to four weeksfollowing transplantation. Transcription of IFN-γ, IL-1β, and TNF-αreached a maximum two to three weeks after engraftment with changes inIFN-γ transcription closely correlating to those changes observed inIL-1β and TNF-α mRNA production.

Example 2 Generation of Synovial T Cell Lines and Adoptive TransferExperiments

[0071] Small pieces (about 10-30 cubic mm) of synovial tissue obtainedfrom RA patients were cultured in 24-well cell culture plates (Costar,Cambridge, Mass.) in RPMI 1640 supplemented with 10% FCS (SummitBiotechnology, Fort Collins, Colo.) and 20 IU/mL of rhIL-2 (Cetus,Emeryville, Calif.). Established T cell lines were maintained by weeklypolyclonal restimulation with immobilized anti-CD3 antibodies and 20IU/mL IL-2. CD4⁺ and CD8⁺ T cell lines were established from synovialtissue-derived T cells by sorting for CD3⁺/CD4⁺ or CD3⁺/CD8⁺ cells on aFACSVantage (antibodies and equipment from Becton DickinsonImmunocytometry Systems, San Jose, Calif.).

[0072] In adoptive transfer experiments, mice were implanted with humansynovial tissue as described in Example 1. Two weeks later, the micewere injected i.p. with 5×10⁷ unsorted T cells, 2.5×10⁷ purified CD4⁺ Tcells, or 2.5×10⁷ purified CD8⁺ T cells expanded from nonimplantedautologous synovial tissue. Control mice received injections with mediumalone. One week after the adoptive transfer, the human synovial tissuewas harvested and analyzed. In selected experiments, human synovium-SCIDmouse chimeras were adoptively transferred with autologous T cellsfourteen days after implantation and were injected i.p. with 250 μgrabbit anti-human IL-16 or normal rabbit IgG (both from Pepro Tech,Rocky Hill, N.J.) on days 17 and 18 following implantation. In theseexperiments, the synovial tissue was harvested on day 22. To assess thefunction of adoptively transferred synovial T cells, the in situtranscription of IFN-γ, IL-1β, and TNF-α in the engrafted synovialtissue was semiquantified by PCR (expressed as transcript numbers afterbeing normalized to the number of β-actin transcripts), and theproduction of cytokine polypeptides in the tissue was examined byimmunohistochemistry.

[0073] PCR-ELISA was used to determine in situ cytokine transcription intissue. Briefly, total RNA was extracted from synovial tissue using acommercially available reagent (Trizol; Life Technologies, Grand Island,N.Y.), and cDNA was synthesized using oligo dT and AMV reversetranscriptase (both obtained from Boehringer Mannheim, Indianapolis,Ind.). cDNA from synovial tissue was adjusted to contain equal numbersof β-actin transcripts. Adjusted cDNA was amplified by PCR withcytokine-specific primers under nonsaturating conditions in parallelwith a standard containing a known number of cytokine sequences.Amplified products were labeled with digoxygenin 11-dUTP (BoehringerMannheim) and then semiquantified in a liquid hybridization assay withbiotinylated internal probes using an ELISA system (BoehringerMannheim). The labeled PCR products were hybridized for two hours with200 ng/mL probe at 42° C. for β-actin, IFN-γ, TGF-β1, and TNF-α, and at55° C. for IL-1β, IL-10 and IL-16. Details of the assay and sequences ofthe primers and probes have been published (Klimiuk P A et al., Am. J.Pathol. 151:1311-1319 (1997); Weyand C M, et al., Arthritis Rheum.40:19-26 (1997); and Brack A et al., J. Clin. Invest. 99:2842-2850(1997)) except for IL-16 (primers: 5′-AAG CTG ACT CCA GAG CCA TGC C-3′(SEQ ID NO:1) and 5′-TCA GCA TGT CCT GCC TAG G-3′ (SEQ ID NO:2); probe:5′-GGC ACT GCC TGA TGG ACC TGT CAC G-3′ (SEQ ID NO:3)). Hybrids wereimmobilized on streptavidin-coated microtiter plates and, after washing,were detected with a peroxidase-conjugated anti-digoxygenin antibody.The plates were developed by a color reaction using ABTS(2,2′-Azino-di-[3-ethylbenzthiazoline sulfonate, (Cooper S M et al.,Arthritis Rheum. 34:537-546 (1991)),] diammonium salt) substrate andquantified using a kinetic microplate reader (Molecular Devices,Sunnyvale, Calif.). The number of cytokine-specific sequences wasdetermined by interpolation on a standard curve and was expressed as thenumber of cytokine sequences per 2×10⁶ β-actin sequences (Weyand C M, etal., Arthritis Rheum. 40:19-26 (1997)).

[0074] The following antibodies were used for immunohistochemistry:mouse anti-CD3 mAb (1:100; Becton Dickinson), mouse anti-CD8 mAb (1:5;Becton Dickinson), mouse anti-IFN-γ mAb (1:100; Genzyme Diagnostics,Cambridge, Mass.), rabbit anti-TNF-α mAb (1:250; Genzyme Diagnostics),polyclonal rabbit anti-IL-16 Ab (1:50; PeproTech, Rocky Hill, N.Y.),mouse anti-CD68 mAb (1:250; Dako, Carpinteria, Calif.), biotinylatedpolyclonal rabbit anti-mouse Ig Ab (1:300; Dako), and polyclonalbiotinylated swine anti-rabbit Ig Ab (1:300; Dako). Briefly, synovialtissues embedded in OCT compound were cut into 5 μm sections, mountedonto gel coated slides (Superfrost/Plus, Fisher Scientific, Pittsburgh,Pa.), and dried in a 37° C. desiccator. The slides were then stored at−70° C. Before staining, slides were fixed in acetone for ten minutes,air dried, and fixed in 1% paraformaldehyde/EDTA, pH 7.2, for threeminutes. Endogenous peroxidase was blocked with 0.3% H₂O₂ in 0.1% sodiumazide. Nonspecific binding was blocked with 5% normal goat serum (LifeTechnologies) for 15 minutes. Sections were stained with primaryantibody for 30 minutes at room temperature. After incubation with theappropriate biotinylated secondary reagent, slides were developed witheither streptavidin-peroxidase, 1:250 (Dako), and 3,3′-diaminobenzidinetetrahydrochloride (DAB; Sigma Chemical, St. Louis, Mo.), or theVectaStain ABC-AP kit and alkaline phosphatase substrate kit-l (VectorLaboratories, Burlingame, Calif.). For two-color immunohistochemistry,slides were then washed in 0.5% Triton X-100/PBS for ten minutes.Nonspecific binding was blocked for 15 minutes with 5% normal goatserum, and the sections were stained with a polyclonal rabbit anti-IL-16antibody for one hour at room temperature. After incubation withbiotinylated swine anti-rabbit antibody, slides were developed with theVectaStain ABC-CP kit. Negative controls without primary antibody wereprocessed in parallel. Sections were counter stained with hematoxylin,and permanently mounted in Cytoseal-280 (Stephens Scientific, Riverdale,N.J.).

[0075] Transfer of unsorted tissue-derived T cells had a profound effecton the functional activity of T cells and macrophages in the humangraft. Injection of autologous T cells consistently resulted in asignificant (p=0.03) suppression of IFN-γ mRNA to ten percent of controllevels (FIG. 2). In grafts from control animals, the median tissueconcentration was 492 IFN-γ transcripts whereas only 52 copies weredetected after adoptive transfer. The transferred cells not onlyinhibited IFN-γ production, but they also affected IL-1β and TNF-αproduction. Median tissue concentrations of IL-1β were reduced from2,974 to 304 transcripts (p=0.03), and TNF-α-specific sequences reducedfrom 17,301 to 1,454 copies (p=0.03).

[0076] To identify the cell subset capable of reducing cytokineproduction in inflamed synovial tissue, T cell lines were sorted intoCD4⁺ and CD8⁺ T cell lines, and cells from each of the subsets wereadoptively transferred. Following injection of 2.5×10⁷ autologous CD4⁺ Tcells, synthesis of IFN-γ and TNF-α mRNA in human synovial tissue wasessentially unchanged (FIG. 3). Conversely, transfer of 2.5×10⁷autologous synovial CD8⁺ T cells induced profound inhibition of IFN-γ aswell as TNF-α transcription (FIG. 3). Control grafts contained a mean of827 IFN-γ transcripts, whereas the mean copy number was reduced to 21 insynovial tissue retrieved from animals injected with CD8⁺ T cells(p=0.10). Similarly, minimal TNF-α mRNA was detectable after transfer ofCD8⁺ T cells (56 copies), whereas control tissues expressed a mean of14,867 copies (p=0.03). In addition, the T cell lines containing CD8⁺ Tcells reduced the transcription of these cytokines to levels similar tothe levels detected in fresh human synovial tissue prior toimplantation. Similar results were obtained following intravenousadministration of autologous sorted and unsorted T cells. These resultsindicate that CD8⁺ T cells can accumulate within the implanted humansynovial tissue after adoptive transfer into the blood stream orperitoneal cavity.

[0077] Immunohistochemical studies of tissue retrieved from humansynovium-SCID mouse chimeras demonstrated that the cellularity of theinfiltrate was not influenced by the adoptively transferred T cells.Tissue sections were stained with anti-CD3 and anti-CD68 monoclonalantibodies and the numbers of T cells and macrophages per section werecounted. The results were generated from three experiments usingsynovial tissue from three patients. About 60-70 CD3⁺ T cells and 50-70CD⁶⁸⁺ macrophages per high power field were present in control tissue(FIG. 4). After adoptive transfer of unsorted synovial T cells or sortedCD4⁺ or CD8⁺ T cells, the number of tissue-infiltrating CD3⁺ T cells andCD68⁺ macrophages did not change. The number of IFN-γ- andTNF-α-producing cells, however, was reduced by about 70 percent aftertransferring CD8⁺ T cells. These results indicate that synovial CD8⁺ Tcells downregulate inflammatory activity in rheumatoid synovitis. Theseresults also indicate that the downregulation of T cell and macrophagefunction is not related to a cytotoxic activity of CD8⁺ T cells since nocell depletion was observed within synovial lesions.

[0078] To determine if the inhibitory activity of adoptively transferredCD8⁺ T cells was related to the production of a cytokine, expression ofIL-16 as well as putative anti-inflammatory cytokines (e.g., IL-4,IL-10, and TGF-β1) in synovial tissue harvested from animals injectedwith medium only, unsorted synovial T cells, sorted CD4⁺ T cells, orsorted CD8⁺ T cells was compared in two separate experiments. Briefly,human tissue grafts were collected from human synovium-SCID mousechimeras following adoptive transfer, and cDNA prepared so that theproduction of IL-4, IL-10, IL-16, and TGF-β1 mRNA transcripts could bemeasured. IL-4 transcripts were not detectable in any harvested tissue.IL-10 transcripts were present at slightly varying amounts without aclear correlation with the type of transferred cell (Table I). However,IL-16 and TGF-β1 transcription inversely correlated with IFN-γ, IL-1β,and TNF-α mRNA production (Table I, and FIGS. 2 and 3). IL-16transcripts were present in low numbers in control grafts (374 and 461copies), but this number increased five-fold upon transfer of eitherunsorted autologous synovium-derived T cells (1910 and 2573 copies) orsorted CD8⁺ T cells (1564 and 2069 copies). Similar results wereobtained for TGF-1, which revealed a ten-fold increase in transcriptnumber after adoptive transfer of unsorted autologous synovium-derived Tcells or sorted CD8⁺ T cells. Transfer of sorted CD4⁺ T cells did notalter synthesis of either IL-16 or TGF-p 1. These results indicated thatIL-16 and TGF-β1 may mediate the downregulatory activity of synovialCD8⁺ T cells. TABLE I Level of cytokine production by human synovialtissue harvested from human synovium-SCID mouse chimeras after adoptivetransfer. Adoptive transfer Medium Unsorted CD4⁺ CD8⁺ Only T cells Tcells T cells Experi- IL-10 1437 ± 131* 1303 ± 139  938 ± 28 1764 ± 333ment1 TGF-β1  597 ± 72 7915 ± 209 1045 ± 51 4856 ± 183 IL-16  461 ± 602573 ± 544  694 ± 14 2069 ± 510 Experi- IL-10  391 ± 14  395 ± 70  202 ±20  357 ± 22 ment 2 TGF-β1  145 ± 23 9840 ± 854  631 ± 3039 ± 81  107IL-16  374 ± 170 1910 ± 71 427 ± 68 1564 ± 375

[0079] Anti-IL-16 antibody was used to determine if theimmunosuppressive effect of adoptively transferred synovial T cells wasat least in part related to IL-16 release. Three days after adoptivetransfer of 5×10⁷ unsorted autologous T cells, human synovium-SCID mousechimeras were treated with two consecutive daily doses of anti-IL-16antibody (250 μg for each dose). Control mice received two consecutivedaily doses of normal rabbit immunoglobulin (250 μg for each dose). Fourdays after antibody treatment, the human synovial tissue was harvestedand analyzed. Adoptive transfer of unsorted tissue-derived T cellsreduced transcription of IL-1β and IFN-γ to about 15 percent ofuntreated controls. In three separate experiments, administration ofanti-IL-16 antibody partially reversed the T cell-mediated suppression.IFN-γ transcription was higher in human synovial tissue harvested fromanimals receiving anti-IL-16 antibody treatment after adoptive transfercompared to tissue harvested from animals not receiving the anti-IL-16antibody (FIG. 5). In parallel, administration of anti-IL-16 antibodyincreased the number of IL-1β transcripts with respect to the levelobserved in tissue harvested from animals not receiving the anti-IL-16antibody treatment. Anti-IL-16 antibody treatment did not restoremaximal synthesis of either of these pro-inflammatory mediators,suggesting either incomplete neutralization of tissue IL-16 or acontribution of another mediator, e.g. TGF-β1, in the suppression ofIFN-γ and IL-1β within tissue.

[0080] To determine if tissue-infiltrating T cells in rheumatoidsynovitis spontaneously synthesized IL-16, immunohistochemical studieswere performed. Synovial tissue sections from six RA patients werestained with anti-IL-16 and T cell specific antibodies. IL-16-positive Tcells were present in all tissues. By two-color immunohistochemistry,the phenotype of IL-16-producing T cells in the synovium was determinedto be predominantly CD8⁺ (FIG. 6). IL-16 was found in 69-93 percent ofall tissue-infiltrating CD8⁺ T cells. In fact, CD8⁺ T cells were thepredominant source of IL-16 in the synovium. Only 6-19 percent of allsynovial cells producing IL-16 were negative for CD8. The small fractionof CD8⁻/IL-16⁺ cells in synovial tissue included T cells as well asnon-T cells, which were probably synoviocytes. There was a tendency forIL-16-producing CD8⁺ T cells to be arranged in clusters and to begrouped in areas of T cell enrichment. No particular spatialrelationship between CD8⁺/IL-16⁺ T cells and TNF-α- and IL-β-producingCD68⁺ cells was detected. The total number of CD8⁺ T cells contributingto the synovial infiltrates varied, suggesting that heterogeneity inrheumatoid synovitis is related to diversity in IL-16-mediatedimmunoregulation.

Example 3 Treating Inflammation with Recombinant Human IL-16

[0081] Human synovium-SCID mouse chimeras were treated with daily i.p.injections of either buffer only or recombinant human IL-16 (rhIL-16;PeproTech) starting on day seven after tissue implantation. Two doses ofrhIL-16 (500 ng and 1000 ng per injection) and two treatment durations(10 and 14 days) were tested. For each cytokine dose and each treatmentduration, four independent human synovium-SCID mouse chimeras werestudied. Implanted tissues were harvested on day 17 for animals treated10 days, and day 21 for animals treated 14 days. Each harvested tissuewas analyzed by immunohistochemistry as well as by PCR-ELISA.

[0082] Treatment with IL-16 markedly reduced in situ transcription ofIFN-γ, IL-1β, and TNF-α (FIG. 7). Reduced transcription of all threepro-inflammatory cytokines was apparent after ten days of treatment withthe low dose of 500 ng rhIL-16. The high dose of rhIL-16 (1000 ng) wasslightly more effective. IFN-γ, IL-1β, and TNF-α transcripts were alldownregulated to about 50 percent of the levels observed in grafts fromcontrol mice. The inhibition was significant for all threepro-inflammatory cytokines for both doses and for both treatmentdurations. Notably, the inhibitory effect of rhIL-16 treatment oncytokine transcription was not a generalized phenomenon. Levels ofTGF-β1 and IL-10 mRNA were unaffected by the injection of exogenousrhIL-16. These results indicate that the increased production of TGF-β1observed after adoptive transfer of CD8⁺ T cells was not a directconsequence of IL-16 release (FIG. 7).

[0083] To evaluate the effects of exogenous rhIL-16 on the synoviallesions, tissue sections from synovial tissue samples harvested fromrhIL-16 treated animals were analyzed. Following treatment with rhIL-16,the microanatomy of the synovial infiltrates was maintained. Inaddition, the number of tissue-infiltrating CD3⁺ T cells and CD68⁺macrophages was conserved (FIG. 8). Injection of rhIL-16, however,markedly changed the functional activity of IFN-γ- and TNF-α-producingcells. Specifically, rhIL-16 treatment of human synovium-SCID mousechimeras almost completely reduced IFN-γ and TNF-α production by thecells present within inflammatory lesions.

[0084] Taken together, the data presented herein indicates that theparadoxical functional silence of tissue infiltrating T cells in RAresults from in situ inhibition of cellular function byanti-inflammatory mechanisms mediated in part by IL-16. In other words,physiologic regulatory pathways are in place that can suppressinflammatory synovitis. In fact, the in vivo results presented hereinindicate that boosting CD8-directed immunosuppression could eradicatesynovial inflammation.

OTHER EMBODIMENTS

[0085] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for treating an inflammatory disease,said method comprising: a) identifying a host in need of ananti-inflammatory treatment, wherein said host has inflamed tissue, andb) administering a pharmaceutically effective amount of an IL-16polypeptide or an IL-16-mimicking molecule to said host under conditionssuch that the expression of an inflammatory cytokine in the region ofsaid inflamed tissue is reduced.
 2. The method of claim 1, wherein saidinflammatory disease is rheumatoid arthritis.
 3. The method of claim 1,wherein said IL-16-mimicking molecule comprises a recombinant HIV gp120polypeptide.
 4. The method of claim 1, wherein said method comprisesadministering, to said host, a polypeptide selected from the groupconsisting of TGF-β1, IL-4 and IL-10.
 5. The method of claim 1, whereinsaid inflammatory cytokine comprises a cytokine selected from the groupconsisting of IFN-γ, IL-1β, and TNF-α.
 6. The method of claim 1, whereinsaid IL-16 polypeptide is recombinant human IL-16.
 7. The method ofclaim 1, wherein said host is a mammal.
 8. The method of claim 7,wherein said mammal is a human.
 9. A method of treating an inflammatorydisease in a host, said method comprising providing nucleic acid to saidhost, wherein said nucleic acid encodes an IL-16 polypeptide and whereinsaid host expresses said IL-16 polypeptide from said nucleic acid suchthat the expression of an inflammatory cytokine is reduced in said host.10. The method of claim 9, wherein said inflammatory disease isrheumatoid arthritis.
 11. The method of claim 9, wherein said methodcomprises providing a second nucleic acid to said host, wherein saidsecond nucleic acid encodes an immunosuppressive cytokine.
 12. Themethod of claim 11, wherein said immunosuppressive cytokine comprises acytokine selected from the group consisting of TGF-β1, IL-4, and IL-10.13. The method of claim 9, wherein said inflammatory cytokine comprisesa cytokine selected from the group consisting of IFN-γ, IL-1β, andTNF-α.
 14. The method of claim 9, wherein said IL-16 polypeptide ishuman IL-16.
 15. The method of claim 9, wherein said host is a mammal.16. The method of claim 15, wherein said mammal is a human.
 17. A methodof treating an inflammatory disease in a host, said method comprisingadministering, to said host, cells that reduce the expression of aninflammatory cytokine.
 18. The method of claim 17, wherein saidinflammatory disease is rheumatoid arthritis.
 19. The method of claim17, wherein said cells are synovial tissue-derived CD8⁺ T cells.
 20. Themethod of claim 17, wherein said cells express IL-16.
 21. The method ofclaim 20, wherein said IL-16 is human IL-16.
 22. The method of claim 17,wherein said cells express TGF-β1.
 23. The method of claim 17, whereinsaid cells contain exogenous nucleic acid encoding an IL-16 polypeptide.24. The method of claim 17, wherein said cells contain exogenous nucleicacid, wherein said exogenous nucleic acid encodes a polypeptide selectedfrom the group consisting of TGF-β1, IL-4, and IL-10.
 25. The method ofclaim 17, wherein said inflammatory cytokine comprises a cytokineselected for the group consisting of IFN-γ, IL-1β, and TNF-α.
 26. Themethod of claim 17, wherein said host is a human.
 27. A pharmaceuticalcomposition for treating an inflammatory disease in a host, saidcomposition comprising an IL-16 polypeptide or an IL-16-mimickingmolecule and an immunosuppressive cytokine, wherein the administrationof said composition to said host reduces the expression of aninflammatory cytokine in said host.
 28. The composition of claim 27,wherein said inflammatory disease is rheumatoid arthritis.
 29. Thecomposition of claim 27, wherein said IL-16 polypeptide is recombinanthuman IL-16.
 30. The composition of claim 27, wherein saidIL-16-mimicking molecule comprises a recombinant HIV gp120 polypeptide.31. The composition of claim 27, wherein said immunosuppressive cytokinecomprises a cytokine selected from the group consisting of TGF-β1, IL-4,and IL-10.
 32. The composition of claim 27, wherein said inflammatorycytokine comprises a cytokine selected from the group consisting ofIFN-γ, IL-1β, and TNF-α.
 33. The composition of claim 27, wherein saidhost is a mammal.
 34. The composition of claim 33, wherein said mammalis a human.
 35. A non-human animal comprising human synovial tissue. 36.The animal of claim 35, wherein said animal is a murine animal.
 37. Theanimal of claim 35, wherein said animal is immunocompromised.
 38. Theanimal of claim 37, wherein said immunocompromised animal is a SCIDmouse.
 39. The animal of claim 37, wherein said immunocompromised animalis a recombination-activating gene-deficient animal.
 40. The animal ofclaim 35, wherein at least a portion of said human synovial tissue islocated under the skin of said animal.
 41. The animal of claim 35,wherein at least a portion of said human synovial tissue is located inthe back region of said animal.
 42. The animal of claim 35, wherein saidhuman synovial tissue comprises inflamed human synovial tissue.
 43. Theanimal of claim 42, wherein said inflamed human synovial tissue is in adiffuse state of inflammation within said animal.
 44. The animal ofclaim 42, wherein said inflamed human synovial tissue is in a follicularstate of inflammation within said animal.
 45. The animal of claim 35,wherein said human synovial tissue comprises diffusely vascularizedhuman synovial tissue within said animal.
 46. The animal of claim 35,wherein said animal is a NOD/LtSz-Prkdc^(scid)/J mouse comprising humansynovial tissue derived from a rheumatoid arthritis patient.
 47. Amethod for identifying a treatment reagent that reduces inflammation,said method comprising: a) administering a test reagent to a non-humananimal having human synovial tissue, wherein at least a portion of saidhuman synovial tissue is inflamed, and b) determining if saidadministration of said test reagent reduces said inflammation, wherein areduction in inflammation indicates that said test reagent is atreatment reagent.
 48. The method of claim 47, wherein said reduction ininflammation is determined by measuring IFN-γ production by said humansynovial tissue.
 49. An article of manufacture comprising packagingmaterial and an IL-16 polypeptide or IL-16-mimicking molecule, whereinsaid packaging material comprises a label or package insert indicatingthat said IL-16 polypeptide or IL-16-mimicking molecule can beadministered to a host for the purpose of treating an inflammatorydisease.
 50. An article of manufacture comprising packaging material andnucleic acid encoding an IL-16 polypeptide, wherein said packagingmaterial comprises a label or package insert indicating that saidnucleic acid can be administered to a host for the purpose of treatingan inflammatory disease.
 51. An article of manufacture comprisingpackaging material and cells that reduce the expression of aninflammatory cytokine, wherein said packaging material comprises a labelor package insert indicating that said cells can be administered to ahost for the purpose of treating an inflammatory disease.
 52. The use ofan IL-16 polypeptide or an IL-16-mimicking molecule in the manufactureof a medicament for treating an inflammatory disease in a host in needof an anti-inflammatory treatment, wherein said host has inflamedtissue, and wherein administering a pharmaceutically effective amount ofsaid medicament to said host reduces the expression of an inflammatorycytokine in the region of said inflamed tissue.
 53. The use of a nucleicacid in the manufacture of a medicament for treating an inflammatorydisease in a host in need of an anti-inflammatory treatment, whereinsaid nucleic acid encodes an IL-16 polypeptide, wherein said host hasinflamed tissue, and wherein administering said medicament to said hostreduces the expression of an inflammatory cytokine in the region of saidinflamed tissue.
 54. The use of cells in the manufacture of a medicamentfor treating an inflammatory disease in a host in need of ananti-inflammatory treatment, wherein said host has inflamed tissue, andwherein administering said medicament to said host reduces theexpression of an inflammatory cytokine in the region of said inflamedtissue.